Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Non-invasive analysis of the embryonic genome for the development of improved preimplantation genetic testing (PGT) protocols

To characterize cell-free DNA origin and determine whether spent blastocyst medium (SBM) is a suitable template for non-invasive genomic assessment of IV§F–generated embryos. As preliminary phase, the origin of aneuploidy was investigated in human blastocysts by using NGS analysis on multiple TE and ICM biopsies from the same embryo. We concluded that whole chromosome aneuploidies are mostly meiotic in origin and can be targeted from SBM samples. On the contrary, we provide definitive evidence about the preferential mitotic nature of segmental aneuploidies, making them as an unsuitable target for SBM analysis. Next, we have assessed the concordance rate and genotyping accuracy of SBM vs embryonic biopsies under standard genetic and IVF laboratory protocols. Under this setting we have highlighted poor predictive performance of embryonic culture media in defining the genetic and chromosomal constitution of the embryo. In particular, In PGT-M tests, for BF and SBM, 2.9% and 20.8% of all samples, respectively, produced a diagnosis concordant with the corresponding TE (n=2 of 69 and 15 of 72, respectively). In PGT-A tests, BF analysis showed high amplification failure rates (65.2%) and an overall concordance rate of 37.5% among amplified samples. After improving embryo culture conditions and genetic protocols for the analysis of SBM samples, we could obtained concordant chromosome analysis between SBM and their relative TE biopsies as high as 87% (N=115) from day 6 embryos in a prospective pilot multicenter study. With the new optimized strategy, a prospective multicenter trial on 1301 paired TE and SBM sample was performed. Results have confirmed high concordance and reproducibility among the IVF centers involved. Improvement in embryo culture and genetic technologies have shown the possibility to utilize SBM for non-invasive assessment of embryonic genetics by the analysis of cfDNA. Future clinical trials are warranted to assess clinical utility of the novel strategy that holds the potentiality of becoming a milestone achievement in the field of preimplantation genetics.